Myocardial injury following an ischemic insult continues to be a challenge in terms of effectively managing or repairing the damage as well as preventing development of chronic heart failure (HF). A real urgency exists to delineate molecular pathological mechanisms for potential new therapeutic targets. Over the last 15 years we have identified a key role for G protein-coupled receptor (GPCR) kinase-2 (GRK2) in the pathophysiology of injured or stressed myocardium. Inhibition of GRK2 activity in small animal models of HF can improve cardiac function and morphology. This has primarily been done using a peptide inhibitor of GRK2, the bARKct, however, recent studies using cardiomyocyte-specific GRK2 knockout (KO) mice have provided similar beneficial results in ischemic HF. Canonically, GRK2 phosphorylates agonist-activated GPCRs, such as b- adrenergic receptors (bARs) in the heart, triggering the process of desensitization. GRK-mediated phosphorylation causes b-arrestin binding to the receptor, which blocks further G protein signaling. Interestingly, b-arrestins (b-arrestin1 and b-arrestin2 are in the heart) can also initiate intracellular signaling pathways after interacting with desensitized receptors through kinase scaffolding, which is independent of G protein signaling. A goal in this proposal is to discover novel GRK2 dependent mechanisms involved in myocardial ischemic injury and repair processes. We now have data that suggests that beneficial GRK2- dependent mechanisms in the injured heart go beyond bAR signaling and control of contractility and include myocyte survival. In this proposal we will test whether acute and/or chronic cardioprotection supported by lower GRK2 expression and/or activity might be due to events beyond cellular contractile signaling including regeneration targeted through either myocytes or stem/progenitor cells. This proposal will also begin the novel investigation into the role of b-arrestins, acting downstream of GRK activity, in myocardial ischemic injury and repair. This will be done using b-arrestin1 and b-arrestin2 KO mice and cultured myocytes and cardiac stem/progenitor cells. In fact, we have preliminary data that demonstrates b-arrestins play a critical role in bone marrow-derived cardiac stem/progenitor cell growth and function. Our Central Hypothesis is that GRK2 activity and subsequent b-arrestin function play novel roles downstream of GPCR desensitization that are critically involved in the heart's response to ischemic injury including potential repair and regeneration. Studies in this Project will employ both in vivo and in vitro techniques addressing cardiac repair through new myocyte formation and also GRK2-related mechanisms involved in the proliferation and function of bone-marrow- derived and resident cardiac stem cells.